G protein Coupled Receptor (GPCR) signal transduction pathways in the striatum, a major reward- processing nucleus in the brain, play a pivotal role in the development of drug addiction. Many drugs of abuse including opioids and psychostimulants produce their effects by activating GPCRs expressed by striatal neurons. Our long-term goal is to elucidate molecular and cellular mechanisms that regulate signaling of the striatal GPCRs as a necessary prerequisite to understanding events that lead to substance dependence and designing strategies for the therapeutic correction. Increasing evidence suggests that Regulators of G Protein Signaling (RGS) proteins play a crucial role in controlling GPCR pathways implicated in addiction. RGS proteins serve to curb G protein signaling and thus are optimally positioned to naturally counteract excessive activation of GPCRs by drugs of abuse. Small molecule therapeutics targeting RGS proteins are emerging as promising therapeutic strategies. However, the mechanisms of RGS action in regulation of striatal G protein pathways are poorly understood. This proposal is focused on delineating the molecular, cellular and behavioral mechanisms by which key striatal RGS protein: the R7-RGS complex regulates signaling in striatal neurons. During the previous period, we made substantial progress in understanding R7-RGS including solving its high-resolution structure, defining its G protein selectivity and identification of cAMP to be critical second messenger system involved in its effects. These observations relied on a host of innovative tools and approaches we developed to study striatal GPCR signaling with high degree of precision. Based on accumulated data we hypothesize that control of opioid and dopamine receptor signaling via G Protein Gao to the downstream cAMP producing effector, adenylate cyclase by allosterically regulated multi- subunit R7-RGS complexes critically shapes behavioral actions of addictive drugs. This hypothesis will be tested by pursuing three complementary Specific Aims that seek to (1) delineate functional role of RGS - Gao axis in controlling cAMP dynamics, (2) determine structure/functional mechanics of RGS complex organization and (3) probe behavioral and circuit relevance of RGS-Gao in controlling responses to opioids and psychostimulants. The strategy proposed to address these Aims will entail a synergistic combination of genetic, biochemical, and physiological approaches, exploiting the existence of a powerful array of reagents, animal models, and innovative assays that allow examining signaling changes in vivo.